Reverse Phase Protein Arrays (RPPA) represent a sensitive antibody based proteomic approach, which enables simultaneous quantification of the abundance of multiple proteins and post-translational modifications across multiple samples. Key applications of our RPPA platform include, dynamic pathway profiling at the post-translational network level following chemical or genetic perturbation and protein biomarker discovery in clinical and preclinical studies. The Zeptosens and Aushon-2470-Nitrocellulose RPPA platforms integrated with near-infrared detection of antibody binding installed within the CRUK Edinburgh Centre incorporate the latest technical advances in RPPA to provide industry leading sensitivity and precise quantification of changes in protein levels and post-translational modifications. We have validated a portfolio of over 360 antibodies, covering key cancer survival, cell–cycle and canonical signaling pathways that can be used to simultaneously profile multiple pathway responses including activation states of druggable pathways across multiple in vitro or in vivo samples including precise time- and dose-studies. Our participation in leading a Global RPPA Society (Mol Cell Proteomics. 2014 Jul;13(7):1625-43) ensures we remain at the cutting edge of RPPA technology and best practices.

The IGMM RPPA Facility incorporates the following core capabilities:

Automated sample handling.

Contact and non-contact spotting of complex lysates and supernatants under humidified and chilled environmental control to minimise sample loss and ensure high reproducibility of quantitative post-translational analysis.

Full barcode tracking integrated with a dedicated database and data visualization software (ZeptoVIEW 3.1 and Mapix) ensures robust data analysis and a seamless link between clinical or preclinical samples with antibody reagents and data analysis.

Close interaction with the Edinburgh Experimental Cancer Medicine Centre, application of a cryo-controlled beadmill tissue homogenizer (FastPrep-24) and a dedicated post funded by the Cancer Research UK Edinburgh Centre to derive quality assured procedures for sample handling and antibody validation places us in an excellent position for applying RPPA to translational studies.

Forward Phase Antibody Microarrays

Our forward phase antibody microarray platform has been developed using the same cutting edge technology we apply within our RPPA facility. This technology makes use of well validated antibody capture/detection pairs sourced from companies like R and D Systems for the relative quantification of secreted factors associated with inflammation and angiogenesis, e.g. chemokines/cytokines/adipokines. Our platform currently comprises of over 90 validated antibody pairs. Unlike many commercial platforms we do not use detection antibody cocktails, instead favouring an approach whereby only the matching capture and detection antibodies are ever mixed. This reduces non-specific signals that can arise by antibody crosstalk, which is a common cause of false positive results in these assays. Arrays can be ran using samples obtained from cell culture or from in vivo experiments.

Our RPPA and Forward Phase facilities are available to external groups through funded collaboration or fee-for-service contract.

People

Kenneth Macleod

Protein and Antibody Microarray laboratory manager

Dr Bryan Serrels

Nitrocellulose RPPA and Antibody Microarray Lead

Professor Neil Carragher

Zeptosens RPPA lead

Contact

If you wish to conduct an RPPA or Forward Phase study with us please contact Alison Munro:

The University of Edinburgh

The University of Edinburgh is a charitable body, registered in Scotland, with registration number
SC005336, VAT Registration Number GB 592 9507 00, and is acknowledged by the UK authorities as a
“Recognised body” which has been
granted degree awarding powers.